A protein called tip of herpesvirus saimiri associates with Lck in transformed T cells. To investigate the effects of complex formation on cellular signal transduction, we constructed human Jurkat-T cell lines expressing tip. Expression of tip in Jurkat-T cells dramatically suppressed cellular tyrosine phosphorylation and surface expression of lymphocyte antigens. Expression of tip also blocked the induction of tyrosine phosphorylation by anti-CD3 stimulation. The expression of tip in fibroblast cells suppressed the transforming activity of oncogenic F505 Lck. Binding assays showed that the SH3 domain of Lck is sufficient to form a stable complex with tip in vitro. These results demonstrate that tip acts at an early stage of the T cell signal transduction cascade by associating with Lck and downregulating Lck-mediated activation. Inhibition of Lck-mediated signal transduction by tip in T cells appears to be analogous to the inhibition of Lyn/Syk-mediated signal transduction in B cells by LMP2A of the B cell tropic Epstein-Barr virus.